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Automated Functional Antibody Purification

Automated Functional Antibody Purification



PhyTip columns provide high throughput small scale automated purification and enrichment of antibodies from microliter to milliliter samples

PhyTip columns provide high throughput small scale automated purification and enrichment of antibodies from microliter to milliliter samples



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    Automated Functional Antibody Purification Automated Functional Antibody Purification Document Transcript

    • Application Note PhyNexus www.phynexus.comEnabling High-Throughput FunctionalCharacterization of Therapeutic AntibodiesIntroductionThe process of drug discovery over the last 50 years has seen momentous change1. In the 1950’s, lead molecules werefound largely by serendipity through in vivo screening of the chemical diversity available at the time, wheresubsequently identified candidates were quickly moved through full development to market.By the 1980’s, with the implementation of systems research and focused screening, discovery developed into alengthier process involving a deeper understanding of the molecular structures and the associated functionalconsequences that contribute to the affinity and efficacy of a specific drug compound. In addition, advances inmolecular and cell biology allowed for direct study and screening against human receptor targets in morerepresentative and complex environments such as animal cells and tissues.In today’s post-genomic era where development costs are increasingly significant drivers, this process has expandedsignificantly to include target identification and validation, as well as lead generation and optimization. With thegreatest expense involved in a new drug being associated with clinical trials, pressure on the discovery process to identifythe lead candidate with the highest likelihood of success has increased considerably. This requires the ability to makeincreasingly informed decisions about the individual leads at earlier stages.Antibody therapeutics and their developmentOne class of drugs that first appeared on the market in the 1980’s is antibody-based therapeutics. Monoclonalantibodies (“Mabs”) represent a significant growth opportunity as indicated by the present value of therapeuticsalready available on the market (2002 global market of US $5.4B for 11 approved drugs), with hundreds of antibodies invarious phases of clinical trials. In addition, Mabs are known to have a higher likelihood of approval as compared toother drug classes once they enter clinical trials, with approval rates for certain Mab types as high as 26%2. Despite thisclear path to future Mab successes, there is always a need to establish even higher approval rates in less time and withlower costs.1. E. Ratti and D. Trist, “Continuing evolution of the drug discovery process in the pharmaceutical industry”, Pure Appl. Chem., 2001, 73, 67-75.2. J. Reichert and A. Pavlou, “Monoclonal antibodies market”, Nature Reviews – Drug Discovery, 2004, 3, 383-384.
    • Page 2 Enabling high-throughput functional characterization of therapeutic antibodiesIn the last ten years of Mab lead discovery, high-throughput strategies have been implemented to initially screen antibodiesto yield sufficient numbers of high-quality leads that are passed on for optimization prior to preclinical development. Initialscreens typically utilize assays to determine the apparent affinity of the potential antibody lead for a given target antigen; itis usually at lead optimization when numerous functional assays are performed to generate data of greater physiologicalrelevance, thus providing more decision-making power as to which lead compounds should proceed for preclinicaldevelopment.By necessity, this strategy places a great deal of importance on the affinity of antibody-target interactions. While therecertainly must be sufficient affinity for the antibody to bind the target, it has been shown in various contexts that affinity anddrug potency are not well correlated.For example, antibodies that possess too high of an affinity have been shown to achieve poor tumor penetration in vivo dueto the so-called binding site barrier effect3, and that antibodies which successfully penetrate tumors typically have affinitieshundreds to thousands of times lower than the highest affinity antibodies4,5,6,. Issues such as these are highly correlated todrug potency and ultimately the efficacy determined within clinical trials. Therefore, the ability to make an earlier decisionas to successful leads on the basis of selection criteria more closely correlated to potency (i.e., criteria other than affinity)is extremely valuable as it translates into less drug being required for desired efficacy, which consequently leads topotentially lower toxicity due to lower drug loads (thus improving chances for approval), as well as lower cost-of-goodsper dose once the drug is approved. Beyond issues related to potency and lowered toxicity, there are also criteria againstwhich to select antibodies that involve the ability to efficiently and cost-effectively manufacture the therapeutic antibody –namely, the stability of the antibody (both in vivo and ex vivo) as well as its efficiency of expression.Selection across these different criteria of the most promising antibody leads requires the ability to have precise controlover the structural – and hence, functional – aspects of the therapeutic antibody under development. Therapeutic antibodydiscovery benefits from today’s capabilities within molecular and cell biology to exert direct influence over these structure-function relationships. The combination of automation and the ability to create vast numbers of different versions, or“libraries” of a biomolecule provides the ability to fine-tune the structure of biotherapeutics in general (includingantibodies) to the level of individual amino acids as well as their modifications, and hence optimize the potential drug forany number of the criteria described above7,8,9. Furthermore, so as to impose the various selection criteria in the mostbiologically meaningful context, cell-based assays are increasingly utilized as an in vitro mimic of highly complex physiologicalprocesses – thus providing deeper and more descriptive data for each lead, upon which more informed decisions can bemade earlier in the process.3. K. Fujimori, D.G. Covell, J.E. Fletcher and J.N. Weinstein, “Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab’)2,and Fab in tumors”, Cancer Res., 1989, 49, 5656-5663.4. G.P. Adams, R. Schier, A.M. McCall, H.H. Simmons, E.M. Horak, R.K. Alpaugh, J.D. Marks and L.M. Weiner, “High affinity restricts the localization andtumor penetration of single-chain Fv antibody molecules”, Cancer Res., 2001, 61, 4750-4755.5. C.P. Graff, K. Chester, R. Begent and K.D. Wittrup, “Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation-half-time at 37oC”, Protein Eng. Des. Sel., 2004, 17, 293-304.6. L.M. Weiner and P. Carter, “Tunable antibodies”, Nature Biotechnol., 2005, 23, 556-557.7. A.L. Kurtzman, S. Govindarajan, K. Vahle, J.T. Jones, V. Heinrichs and P.A. Patten, “Advances in directed protein evolution by recursive genetic recombi-nation: applications to therapeutic proteins”, Curr. Opin. Biotechnol., 2001, 12, 361-370.
    • Enabling high-throughput functional characterization of therapeutic antibodies Page 3However, cell-based assays require that antibodies screened within it are well purified and enriched once they have beenexpressed, since typical antibody concentrations and contaminants inherent to cell culture completely confound directanalyses. The process for adequately preparing antibodies requires that sufficient quantities of material be scaled up to atleast tens of milliliters, and then processed in a time-consuming and serial manner using expensive chromatographyequipment – all of which precludes taking a cell-based screening approach in the earliest stages. Consequently, cell-basedassays continue to be performed considerably downstream from the initial selection screens, thus relegating the high-valueinformation they provide to the latter stages of drug development and depriving therapeutic antibody developers of thecritical information they need to make increasingly informed decisions earlier in their processes.Obtaining high-value biofunctional information at the earliest stages ofantibody discoveryRecent advances by PhyNexus in the area of miniaturized high-throughput tools for purification, enrichment and desalting ofantibodies and recombinant proteins now enable the implementation of cell-based assays at the earliest stages of leadscreening. By performing high-performance functional protein separations on samples as small as a few hundredmicroliters, it is now possible to obtain more physiologically relevant data from thousands of distinct antibody variants and todo so with complete automation – thus making substantial improvements in return-on-investment by dramaticallyincreasing the decision-making power available at the earliest stages in the antibody discovery process.PhyNexus’ PhyTip® column technology has been developed for high-throughput preparation of antibodies andrecombinant proteins in order to facilitate the process of preparing hundreds to thousands of potential antibody leads thatare ready for cell-based assays without the need for scale-up. These unique columns are designed to operate either on96-at-a-time platforms (such as those from Caliper, Tecan, Perkin-Elmer and Beckman) or on PhyNexus’ automated MEAPersonal Purification System. In either case, the PhyTip columns utilize a unique combination of design and process tomaximize the potential of the enclosed affinity resin to capture the protein of interest. The figures below show the MEAPersonal Purification System and the PhyTip columns.The operational process utilized by the PhyTip columns requires the robotics platform to move to the sample and pass agiven volume over the resin bed at a specific flow rate to obtain the highest performance from the individual steps ofcapture, purification and enrichment. Typical results from a 96-at-a-time platform indicate that >95% purity of fullyfunctional scFvs, Fabs or IgGs can be obtained with exceptionally high yields and enrichment factors in as little as 15minutes for 96x200 µL samples10 or 60 minutes for 96x5 mL samples11.10. J. Lambert, M. Anderson, C. Hanna, L. Jordan, A. Esterman and S. Cohen, “Improved process efficiency with 96-well protein purification andcharacterization”, 15th Annual International Conference on Antibody Engineering, San Diego, CA, 1-3 Dec, 2004.11. K. Kopacz, C. Pazmany, Q. Wu, J. Cosic, A. Nixon and D. Sexton, “Automated purification of phage display-derived antibody sFab fragments”, 10thSociety of Biomolecular Screening Annual Conference, Orlando, FL, 11-15 Sept, 2004.
    • 8. M.A. Poul, B. Becerril, U.B. Nielsen, P. Morisson and J.D. Marks, “Selection of tumor-specific internalizing human Page 4 antibodies from phage libraries”, J. Mol.. Biol., 2000, 301, 1149-1161. Additionally, PhyTip 5K desalting columns used in conjunction with the MEA Personal Purification System allow for completely automated desalting of 96 functional antibodies in less than 45 minutes. These capabilities have allowed various groups’ to successfully enable high-throughput biofunctional screening data to be obtained for a wide range of antibody types and assay formats. For example: • High-throughput cell-based assays of growth factor receptor-mediated Stat5 phosphorylation have been used at Amgen to screen for potent antagonist antibodies derived from phage display12. Individual clones of human scFv recombinant antibodies were expressed in E.coli overnight growths in 96x2 mL deep well plates. 300 µL periplasmic preps were pre- pared for each clone and were individually processed with PhyTip columns containing Ni-NTA resin. • High-throughput flow cytometry has been used at Dyax to perform assays for whole cell-binding IgGs13. Individual clones of human Fab recombinant antibodies were initially selected by standard panning procedures, individual clones were picked, reformatted into IgG structures and expressed as HEK293 transient transfections. 96x6 mL cultures were incubated in 4 separate 24x10 mL deep well plates and were redistributed into 96x2 mL deep well plates, which were then processed with PhyTip columns containing Protein A resin. This same group also uses PhyTip columns to perform high-throughput surface plasmon resonance (SPR) array screening of recombinant antibody kinetics14. • High-throughput target identification, target validation and lead screening have been streamlined and automated at Raven by immobilizing lead antibodies on PhyTip columns and performing a range of assays with them – from IP and CoIP to identify targets, ELISAs of whole cells and cell lysates to confirm targets, and immunohistochemistry (IHC) of cell and tissue arrays as part of lead screening and target validation. This process ensures that only the most well-qualified leads are pursued – thus leading to substantial improvements in process efficiency, more focused lead development efforts and overall cost savings15. Conclusions Antibody-based therapeutics have begun to prove themselves as a powerful class of drugs. The intrinsic ability to fine-tune antibody function with respect to the disease target, the proven track record of antibody therapeutics within clinical trials, and the already burgeoning value of the antibody therapeutic market indicate a bright future for this class of therapeutics. Nevertheless, a constant push will always exist to ensure even greater efficacy and improved approval rates so as to maximize return-on-investment for those organizations involved in antibody therapeutics. An essential part of meeting these goals is to insure that resources are directed towards implementing the most efficient processes to developing those antibody leads that are the most relevant and have the highest likelihood of success; to do so requires obtaining as much physiologically relevant data as early as possible so that key decisions are made before significant resources are committed. PhyNexus’ PhyTip column technology for purification and desalting represents a critical component in the process of obtaining this decision-making power at the earliest stages of antibody discovery and development. 12. C. Hanna, D. Gjerde, L. Nguyen, D. Burge, J. Lambert and T. Arvedson, “Novel strategies for assaying recombinant antibody function with high- throughput cell-based assays”, 7th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA, 16-20 May, 2005. 13. K. Kopacz, C. Pazmany, L. Huang, R. van Hegelsom, H. Pieters, N. Frans, M. Nguyen, A. Gorman, I. Roy, Q. Chang and A. Nixon, “Automated purification of phage display-derived antibodies for downstream analysis”, 7th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA, 16-20 May, 2005. 14. J. Lambert, C. Hanna, U. Banik, D. Sexton, K. Kopacz and S. Wiltshire, “A novel approach to high-throughput monoclonal and recombinant antibody enrichment and characterization”, 6th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA, 26-29 April, 2004. 15. C.Hanna, D. Gjerde, J. Lambert, T. Liang, J. Whelan, C. Fieger and I. Ni, “Automated enhancement and streamlining of therapeutic antibody discovery through the application of micro-scale high-performance protein separation technology”, 20th Annual Drug Discovery Technology, Boston, MA, 8-11 August, 2005.Copyright © 2005, PhyNexus, Inc., All Rights ReservedPSL # 90-00-01 www.phynexus.com